Phosphonic acid derivatives and use thereof

ABSTRACT

A phosphonic acid derivative compound represented by formula  I ! or a pharmaceutically acceptable salt thereof: ##STR1## wherein R 1 , R 2  and R 3  each represent hydrocarbon groups which may be substituted, except cases in which (1) R 2  is unsubstituted methyl, ( 2 ) R 3  is an unsubstituted hydrocarbon group having 1 to 3 carbon atoms, and (3) R 1  is benzyloxycarbonylaminomethyl, R 2  is isobutyl and R 3  is isobutyl or phenylmethyl, which has endothelin-converting enzyme inhibiting activity and is useful as pharmaceutical drugs such as therapeutic agents for hypertension, cardiac or cerebral circulatory diseases and renal diseases. 
     This is a Reissue of a Patent which was the subject of a Reexamination Certificate No. B1 5,330,978, dated Jun. 18, 1996, Request No. 90/00400, Oct. 18, 1995.

BACKGROUND OF THE INVENTION

The present invention relates to novel phosphonic acid derivativeshaving endothelin-converting enzyme inhibiting activity, and is furtherdirected to methods for producing the same and their use.

Endothelin is a vasoconstrictive peptide composed of 21 amino acidresidues which was isolated from the culture supernatant of theendothelin cells of porcine aortas and whose structure was determined byYanagisawa et al. Yanagisawa et al., Nature, 332, 411-415 (1988)!. Fromthe research on genes coding for endothelin, as the biosyntheticmechanism of endothelin, endothelin was deduced to be biosynthesizedfrom an endothelin precursor through big endothelin (ibid.). Thesubsequent studies have revealed the presence of enzymes for convertingbig endothelin to endothelin (endothelin-converting enzymes) Ikekawa etal., Biochem. Biophys. Res. Commu., 171, 669-675 (1990); and Okada etal., ibid., 171, 1192-1198 (1990)!.

Endothelin has vasopressor activity, so that it is anticipated to be anintrinsic factor responsible for the control of circulatory systems anddeduced to be related to hypertension, cardiac or cerebral circulatorydiseases and renal diseases. Inhibitors for the endothelin-convertingenzymes are potential therapeutic drugs for these diseases. At present,however, no endothelin-converting enzyme inhibiting substances otherthan phosphoramidon are reported.

SUMMARY OF THE INVENTION

As a result of intensive investigation, the present inventors creatednovel compounds of the present invention having endothelin-convertingenzyme inhibiting activity.

According to the present invention, there is provided a phosphonic acidderivative compound represented by formula I! or a pharmaceuticallyacceptable salt thereof: ##STR2## wherein R₁, R₂ and R₃ each representhydrocarbon groups which may be substituted, except cases in which (1)R₂ is unsubstituted methyl, ( 2 ) R₃ is an unsubstituted hydrocarbongroup having 1 to 3 carbon atoms, and (3) R₁ isbenzyloxycarbonylaminomethyl, R₂ is isobutyl and R₃ is isobutyl orphenylmethyl. The present invention further provides a method forproducing the same and use thereof.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

When amino acids and peptides are indicated by abbreviations in thisspecification, they are based on the abbreviations adopted by theIUPAC-IUB Commission on Biochemical Nomenclature or commonly used in theart. Examples thereof are as follows:

Val: Valine

Nva: Norvaline

Leu: leucine

Ile: Isoleucine

Nle: Norleucine

Met: Methionine

Cha: Cyclohexylalanine

Phe: Phenylalanine

Trp: Tryptophan

Protective groups and reagents frequently used in this specification areindicated by the following abbreviations:

Boc: t-Butoxycarbonyl

Bzl: Benzyl

HONB: N-hydroxy-5-norbornene-2,3-dicarboximide

DCC: N,N'-dicyclohexylcarbodiimide

DCHA: N,N'-dicyclohexylamine

In the compound of the present invention represented by formula I!, R₁,R₂ and R₃ each represent hydrocarbon groups which may be substituted,except cases in which (1) R₂ is unsubstituted methyl, (2) R₃ is anunsubstituted hydrocarbon group having 1 to 3 carbon atoms, and (3) R₁is benzyloxycarbonylaminomethyl, R₂ is isobutyl and R₃ is isobutyl orphenylmethyl.

As the above-mentioned hydrocarbon group represented by R₁, an alkylgroup, a cycloalkyl group or an aralkyl group is preferred. As the alkylgroup, a straight chain or branched chain alkyl group having 1 to 12carbon atoms is preferred. Examples thereof include methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,isoamyl, tert-amyl, n-hexyl, n-octyl, n-decyl and n-dodecyl. These alkylgroups may be substituted. The substituent groups include cycloalkylsuch as cyclopropyl, cyclopentyl, cyclohexyl and cycloheptyl; halogensuch as fluoro, chloro and bromo; hydroxy which may be protected; alkoxysuch as methoxy and ethoxy; ketone, amino which may be protected; andsubstituted amino. The substituted alkyl groups include, for example,cyclohexylmethyl, 2-cyclohexylethyl, 2-fluoroethyl, 2-chloroethyl,3-chloropropyl, 2-hydroxyethyl, 2-methoxyethyl and 2-aminoethyl. As thecycloalkyl group, a 5-, 6- or 7-membered alicyclic alkyl group ispreferred. Examples thereof include cyclopentyl, cyclohexyl andcycloheptyl. These cycloalkyl groups may be substituted. The substituentgroups include lower alkyl such as methyl, ethyl and n-propyl; halogensuch as fluoro, chloro and bromo; hydroxy which may be protected; alkoxysuch as methoxy and ethoxy; ketone, amino which may be protected; andsubstituted amino which may be protected. The substituted cycloalkylgroups include, for example, 4-methylcyclohexyl, 4-chlorocyclo-hexyl,4-hydroxycyclohexyl and 4-methoxycyclohexyl. As the aralkyl group, analkyl group having 1 to 5 carbon atoms substituted by an aromatichydrocarbon group having 6 to 12 carbon atoms is preferred. Examplesthereof include phenylmethyl (benzyl), 2-phenylethyl (phenethyl),1-naphthylmethyl, 2-naphthylmethyl, 2-(1-naphthyl)ethyl,2-(2-naphthyl)ethyl and 3-phenylpropyl. These aralkyl groups may besubstituted. The substituent groups include lower alkyl such as methyl,ethyl and n-propyl; cycloalkyl such as cyclopentyl and cyclohexyl;halogen such as fluoro, chloro and bromo; hydroxy which may beprotected; and alkoxy such as methoxy and ethoxy. The substitutedaralkyl groups include, for example, 4-methylphenylmethyl,2-(4-methylphenyl)ethyl, 4-fluorophenylmethyl, 2-(4-chlorophenyl)ethyland 2-(4-methoxyphenyl)ethyl. Preferred examples of R₁ include isoamyl,cyclohexylmethyl, 2-phenylethyl, 1-naphthylmethyl, 2-naphthylmethyl,2-(1-naphthyl)ethyl and 2-(2-naphthyl)ethyl.

As the above-mentioned hydrocarbon group represented by R₂, an alkylgroup, a cycloalkyl group or an aralkyl group is preferred. As the alkylgroup, a straight chain or branched chain alkyl group having 1 to 8carbon atoms is preferred. Examples thereof include methyl (exceptunsubstituted methyl), ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, n-pentyl, isoamyl, tert-amyl, n-hexyl andn-octyl. These lower alkyl groups may be substituted. The substituentgroups include cycloalkyl such as cyclopentyl, cyclohexyl andcycloheptyl; halogen such as fluoro, chloro and bromo; hydroxy which maybe protected; mercapto which may be protected; alkoxy such as methoxyand ethoxy; alkylthio such as methylthio and ethylthio; amino which maybe protected; substituted amino which may be protected; guanidino whichmay be protected; carboxyl which may be protected; carbamoyl; ketone;and heterocyclic groups, wherein heterocyclic groups mean groupsobtained by eliminating hydrogen atoms bound to carbon atoms ofmonocyclic to tricyclic heterocycles containing 1 to 3 nitrogen atomsand/or oxygen atoms and/or sulfur atoms as ring constituent atoms otherthan carbon atoms, for example, 2-pyridyl, 3-pyridyl, 4-pyridyl,2-pyridazinyl, 3-furyl, 3-thienyl, 2-indolyl, 3-indolyl, 4-thiazolyl,4-imidazolyl, benzofuryl and benzothienyl. The substituted lower alkylgroups include, for example, cyclohexylmethyl, 2-cyclo-hexylethyl,2-fluoroethyl, 2-chloroethyl, 3-chloropropyl, hydroxymethyl,2-hydroxyethyl, 2-methoxyethyl, 2-amino-ethyl, 4-aminobutyl,3-guanidinopropyl, carbamoylmethyl, 2-carbamoylethyl, mercaptomethyl,carboxymethyl, 2-carboxyethyl, 4-imidazolylmethyl and 3-indolylmethyl.As the cycloalkyl group, a 5-, 6 or 7-membered alicyclic alkyl group ispreferred. Examples thereof include cyclopentyl, cyclohexyl andcycloheptyl. These cycloalkyl groups may be substituted. The substituentgroups include lower alkyl such as methyl, ethyl and n-propyl; halogensuch as fluoro, chloro and bromo; hydroxy which may be protected; andalkoxy such as methoxy and ethoxy. The substituted cycloalkyl groupsinclude, for example, 4-methylcyclohexyl, 4-chlorocyclohexyl,4-hydroxycyclohexyl and 4-methoxycyclohexyl. As the aralkyl group, analkyl group having 1 to 5 carbon atoms substituted by an aromatichydrocarbon group having 6 to 12 carbon atoms is preferred. Examplesthereof include phenylmethyl, 1-naphthylmethyl, 2-phenylethyl,2-naphthylmethyl, 2-(1-naphthyl)ethyl, 2-(2-naphthyl)ethyl and3-phenylpropyl. These aralkyl groups may be substituted. The substituentgroups include lower alkyl such as methyl, ethyl and n-propyl;cycloalkyl such as cyclopentyl, cyclohexyl and cycloheptyl; halogen suchas fluoro, chloro and bromo; hydroxy which may be protected; and alkoxysuch as methoxy and ethoxy. The substituted aralkyl groups include, forexample, 4-methylphenylmethyl, 2-(4-methylphenylethyl,4-fluorophenylmethyl, 2-(4-chlorophenyl)ethyl and2-(4-methoxyphenyl)ethyl. Preferred examples of R₂ include n-propyl,isopropyl, isobutyl, sec-butyl, cyclohexylmethyl and benzyl, andisobutyl is particularly preferred among others.

As the above-mentioned hydrocarbon group represented by R₃, an alkylgroup, a cycloalkyl group or an aralkyl group is preferred. As the alkylgroup, a straight chain or branched chain unsubstituted alkyl grouphaving 4 to 8 carbon atoms or a substituted alkyl group having 1 to 8carbon atoms is preferred. Examples of the unsubstituted alkyl groupshaving 4 to 8 carbon atoms include n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, isoamyl, tert-amyl, n-hexyl, isohexyl, n-heptyland n-octyl. The substituent groups of the alkyl groups having 1 to 8carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, isoamyl, tert-amyl, n-hexyl,isohexyl, n-heptyl and n-octyl, include cycloalkyl such as cyclopentyl,cyclohexyl and cycloheptyl; halogen such as fluoro, chloro and bromo;hydroxy which may be protected; mercapto which may be protected; alkoxysuch as methoxy and ethoxy, alkylthio such as methylthio and ethylthio;amino which may be protected; substituted amino which may be protected;guanidino which may be protected; carboxyl which may be protected;carbamoyl; ketone; and heterocyclic groups, wherein heterocyclic groupsmean groups obtained by eliminating hydrogen atoms bound to carbon atomsof monocyclic to tricyclic heterocycles containing 1 to 3 nitrogen atomsand/or oxygen atoms and/or sulfur atoms as ring constituent atoms otherthan carbon atoms, for example, 2-pyridyl, 3-pyridyl, 4-pyridyl,2-pyridazinyl, 3-furyl, 3-thienyl, 2-indolyl, 3-indolyl, 4-thiazolyl,4-imidazolyl, benzofuryl and benzothienyl. The substituted alkyl groupsinclude, for example, cyclohexylmethyl, 2-cyclohexylethyl,2-fluoro-ethyl, 2-chloroethyl, 3-chloropropyl, hydroxymethyl,2-hydroxyethyl, 2-methoxyethyl, 2-aminoethyl, 4-aminobutyl,3-guanidinopropyl, carbamoylmethyl, 2-carbamoylethyl, mercaptomethyl,carboxymethyl, 2-carboxyethyl, 4-imidazolylmethyl and 3-indolylmethyl.As the cycloalkyl group, a 5-, 6- or 7-membered alicyclic alkyl group ispreferred. Examples thereof include cyclopentyl, cyclohexyl andcycloheptyl. These cycloalkyl groups may be substituted. The substituentgroups include lower alkyl such as methyl, ethyl and n-propyl; halogensuch as fluoro, chloro and bromo; hydroxy which may be protected; andalkoxy such as methoxy and ethoxy. The substituted cycloalkyl groupsinclude, for example, 4-methylcyclohexyl, 4-chlorocyclohexyl and4hydroxycyclohexyl. As the aralkyl group, an alkyl group having 1 to 5carbon atoms substituted by an aromatic hydrocarbon group having 6 to 12carbon atoms is preferred. Examples thereof include phenylmethyl,2-phenylethyl, 2-naphthylmethyl, 2-(1-naphthyl)ethyl,2-(2-naphthyl)ethyl and 3-phenylpropyl. These aralkyl groups may besubstituted. The substituent groups include lower alkyl such as methyl,ethyl and n-propyl; cycloalkyl such as cyclopropyl, cyclopentyl,cyclohexyl and cycloheptyl; halogen such as fluoro, chloro and bromo;hydroxy which may be protected; and alkoxy such as methoxy and ethoxy.The substituted aralkyl groups include, for example,4-methylphenylmethyl, 2-(4-methylphenyl)ethyl, 4fluorophenylmethyl,2-(4-chlorophenyl)ethyl and 2-(4-methoxyphenyl)ethyl. Preferred examplesof R₃ include 3-indolylmethyl which may be substituted.

The carbon atoms of the compound of the present invention represented byformula I! to which R₂ and R₃ are bound are all asymmetric carbon atoms.In the present invention, these carbon atoms include all the L-, the D-and the racemic forms.

The compounds of the present invention include a compound having formulaIV! or a pharmaceutically acceptable salt thereof: ##STR3## where R₁ isas defined previously for Compound I! and U and V are amino acidresidues connected to the P-atom at the N-terminus and selectedindependently from substituted or unsubstituted amino acid residues withthe proviso that (1) U is not a residue of Ala, (2) V is not a residueof Ala or Val and (3) when R₁ is benzyloxycarbonylaminomethyl and U is aresidue of Ile, V is not a residue of Ile or Phe. Preferred amino acidresidues for compounds of the invention are derived from the twentycommon amino acids found in proteins. Substituted amino acid residues inaccord with the present invention contain substitutions as definedhereinabove with respect to R₁, R₂ and R₃. Such substituent hydrocarbongroups may also be used as substitutions for amino acid residues offormula IV.

The compounds of the present invention include salts of the compoundrepresented by formula I!. Such salts preferably include an ammoniumsalt, alkali metal salts such as a sodium salt and a potassium salt;alkaline earth metal salts such as a calcium salt and a magnesium salt;and organic base salts such as a pyridine salt and a triethylamine salt.Most preferably, the salts are pharmaceutically acceptable salts orsalts that can be converted to pharmaceutically acceptable salts.

The compound of the present invention represented by formula I!,hereinafter occasionally referred to as compound I! can be produced, forexample, in the following manner. Namely, an organic phosphorus compoundrepresented by formula II!, hereinafter occasionally referred to ascompound II! is reacted with a dipeptide compound represented by formulaIII!, hereinafter occasionally referred to as compound III!, or a saltthereof to prepare a phosphonic acid compound: ##STR4## wherein Arepresents a protected hydroxyl group; X represents a halogen atom; Yrepresents a protected carboxyl group; and R₁ ', R₂ ' and R₃ ' eachrepresent hydrocarbon groups which may be substituted (for example, thegroups listed hereinabove for R₁, R₂ and R₃, respectivley), thehydrocarbon groups being protected with protecting groups if they havegroups required to be protected, except cases in which (1) R₂ ' isunsubstituted methyl, (2) R₃ ' is an unsubstituted hydrocarbon grouphaving 1 to 3 carbon atoms, and (3) R₁ ' isbenzyloxycarbonylaminomethyl, R₂ ' is isobutyl and R₃ ' is isobutyl orphenylmethyl.

The resulting phosphonic acid compound is represented by formula I'! andhereinafter occasionally referred to as compound I'!: ##STR5## whereinA, Y, R₁ ', R₂ ' and R₃ ' have the meanings given above.

This compound is subjected to treatment for eliminating the protectinggroups to obtain compound I!. In some cases, the protecting groups areeliminated concurrently with the reaction of phosphorus compound II!with dipeptide compound III! or the salt thereof, whereby compound I!can be obtained without treatment for eliminating the protecting groups.

"A" of organic phosphorus compound II! is the protected hydroxyl group.As the protecting group, a known protecting group for P-OH is used.Specifically preferred such protecting groups include benzyl and4-methylbenzyl. "X" of organic phosphorus compound II! is halogen, forexample, chloro, bromo or iodo. "Y" of dipeptide compound III! is theprotected carboxyl group. As the protecting group, a known protectinggroup for a carboxyl group is used. Specifically preferred suchprotecting groups include benzyl and 4-methylbenzyl. The salt ofdipeptide compound III! preferably means a salt at the terminal aminogroup. Such salts include salts with inorganic acids such ashydrochloric acid and sulfuric acid; and salts with organic acids suchas acetic acid and p-toluenesulfonic acid.

Described in more detail, this method comprises reacting organicphosphorus compound II! with dipeptide compound III! in an organicsolvent, followed by treatment for eliminating the protecting groups toobtain the, desired compound- I!. It is generally preferred to isolatethe first reaction product, i.e. resulting phosphonic acid compoundII'!, before removing the protecting groups.

The organic solvent is selected from organic solvents which do not reactwith compounds II! and III!. Organic solvents preferably used are ethersolvents such as tetrahydrofuran and dioxane, and halogenatedhydrocarbon solvents such as dichloromethane and dichloroethane.Dichloromethane is most suitable among others. These solvents arepreferably used in the anhydrous state. The amount of the organicsolvent is usually about 10 to 100 ml/mmol of compound III! and morepreferably about 30 to 50 ml/mmol. Compound II! is usually used in thesame amount as that of compound III! or in excess, specifically in anamount of 1 to 2.5 mmol/mmol of compound III! and preferably in anamount of 1 to 1.5 mmol/mmol. The reaction of compound II! with compoundIII! is dehydrohalogenation reaction.

Bases are preferably used to eliminate hydrogen halides. Such bases usedinclude inorganic bases such as sodium hydroxide and potassiumhydroxide; and organic bases such as triethylamine andN-methylmorpholine; and the organic bases are more preferable amongothers. The bases are used in an amount enough to neutralize thehydrogen halides and the salts of compound III!, or in an amount of morethan that. In some cases, liquid organic bases such as pyridine are usedpartly for solvents.

The reaction is conducted under cooling or at room temperature, usuallyat a temperature of about 0° to 25° C. The reaction time is usually 0.5to 2 hours, though it varies depending on the kind of starting compoundand the reaction temperature. After reaction, compound I'! can beisolated from the reaction solutions by combinations of known purifyingtechniques such as solvent extraction, distillation, columnchromatography, liquid chromatography and recrystallization.

Protecting group eliminating reagents may be further added to thereaction Solutions containing compound I'! to produce the desiredcompound I!. The protecting groups of compound I'! can be eliminatedusing known protecting group eliminating reagents, according to knownmethods, although they may vary depending on the kind of protectinggroups as is well known to those skilled in the art After treatment forelimination of the protecting groups, compound I! can be isolated fromthe reaction solutions by combinations of known purifying techniquessuch as solvent extraction, distillation, column chromatography, liquidchromatography and recrystallization. As a matter of course, treatmentfor isolation of compound I'! prior to eliminating the protecting groupstypically makes purification of the desired compound I! easier thanwithout isolation of compound I'!.

Compounds II! and III! used as starting compounds are commerciallyavailable or can be produced by or in accordance with methods known inthe art.

Compound I! of the present invention, including the pharmaceuticallyacceptable salts thereof, are useful as pharmaceutical drugs such astherapeutic agents for hypertension, cardiac or cerebral circulatorydiseases and renal diseases. The pharmaceutically acceptable saltsinclude, for example, an ammonium salt; alkali metal salts such as asodium salt and a potassium salt; alkaline earth metal salts such as acalcium salt and a magnesium salt; and organic base salts such as apyridine salt and a triethylamine salt. As these therapeutic agents,compound I! can be administered in a treatment effective amount orallyor parenterally in the form of a liquid formulation or a solidformulation to mammals such as humans, rabbits, dogs, cats, rats andmice. Usually, it is administered parenterally in the form of a liquidformulation, such as an injection. The dosage varies depending on thetype of disease to be treated, the symptoms of the disease, the objectto which the drug is given and the route of administration. For example,when parenterally given to human adult for treatment of hypertension, itis advantageous that compound I! is given in the form of an injection byintravenous injection in one dose of about 0.01 to 20 mg/kg of weight,preferably about 0.05 to 10 mg/kg about once to 3 times a day. In thecase of other routes and forms of administration, compound I! can alsobe given in a dose similar thereto. The injections include subcutaneousinjections, intracutaneous injections, intramuscular injections and dripinjections, as well as intravenous injections. Such injections areprepared by methods known per se in the art, namely by dissolving,suspending and emulsifying compound I! in sterile aqueous solutions oroily solutions. The aqueous solutions for injection includephysiological saline and isotonic solutions containing glucose or otheradjuvants, and may be used in combination with appropriate solubilizerssuch as alcohols, e.g. ethanol; polyalcohols, e.g. propylene glycol andpolyethylene glycol; and nonionic surface active agents, e.g.Polysolvate 80 and HCO-50. The oily solutions include sesame oil andsoybean oil, and may be used in combination with solubilizers such asbenzyl benzoate and benzyl alcohol. The injections thus prepared areusually packed in appropriate capsules. Conveniently, compound 51 ispackaged in a unit dose in a pharmaceutically acceptable carrier in acapsule or tablet form, or in a vial. The unit does contains an amountof compound I!, as set forth above, for one administration to a patient.Typically, the unit dose will contain from about 0.5. to about 1500 mg,preferably about 2.5 to about 750 mg, of compound I!. Vials can alsoconveniently be packaged for multiple doses for treatment for aprescribed period of time.

Compound I! of the present invention has endothelin-converting enzymeinhibiting activity as shown by the procedures described in the TestExample. Endothelin is a peptide having vasoconstrictive activity, andtherefore compound I! is useful as pharmaceutical drugs such astherapeutic agents for hypertension, cardiac or cerebral circulatorydiseases and renal diseases.

The present invention will be described in more detail with thefollowing test example, reference example and examples. It is understoodof course that these test example, reference example and examples arenot intended to limit the scope of the invention. In the examples, allamino acids other than glycine take the L-form unless otherwisespecified. In the examples, silica gel 60F-254 (Merck) was used asplates of thin layer chromatography, and Rf¹ : chloroform-methanol (9:1)and Rf² : isopropanol-water-concentrated aqueous ammonia (5:2.0.2) wereused as developing solvents.

TEST EXAMPLE

Measurement of Endothelin-Converting Enzyme Inhibiting Activity ofPhosphonic Acid Amide Derivatives

In 1.5-ml centrifugal tube (BIO-BIK), 80 μl of 50 mMbis-Trispropane-hydrochloric acid buffer (pH 7.2) supplemented with 1.0mg/ml bovine serum albumin, 0.1M sodium chloride, 1.0 mMphenylmethanesulfonyl fluoride (PMSF, Wako Pure Cnemical Industries),1.0 μg/ml leupeptin (Peptide Institute Inc.), 1.0 μg/ml chymostatin(Peptide Institute Inc.), 1.0 μg/ml pepstatin A (Peptide InstituteInc.), 1.0 μM E-64 (Peptide Institute Inc.), 1.0 μm thiorphan (Sigma)and 1.0 μM angiotensin-converting enzyme inhibitor CV-5975, Inada etal., Japanese Journal of Pharmacology, 47, 135-141 (1988)!, 5 μl of asample solution to be tested (an aqueous solution or an aqueous solutioncontaining 2% dimethyl sulfoxide) or 5 μl of distilled water for thecontrol plot, and 10 μl of an endothelin-converting enzyme sampleprepared as described in the following reference example were placed.After standing at 37° C. for 30 minutes, 5 μl of a substrate solutionphysiological phosphate buffer (PBS, containing 0.2 g of potassiumchloride, 0.2 g of potassium dihydrogenphosphate, 8.0 g of sodiumchloride and 1.14 g of disodium hydrogenphosphate in 1,000 ml ofdistilled water) supplemented with 1.0×10⁻⁶ M pig big endothelin I(1-39) (Peptide Institute Inc.) and 1.0 mg/ml bovine serum albumin! wasadded thereto to initiate enzyme reaction. After reaction at 37° C. for1 hour, the reaction mixture was boiled in boiling water for 5 minutes,thereby terminating the reaction: The resulting insoluble material wasremoved by centrifugation, and endothelin I (1-21) produced by theenzyme reaction contained in 10 μl of the supernatant was quantified bythe endothelin I-specific sandwich enzyme immunoassay method alreadyestablished Suzuki et al., J. Immuno. Meth., 188, 245-250 (1989)!, andcompared with the amount of endothelin produced in the control plot toevaluate the enzyme inhibiting activity. The endothelin-convertingenzyme inhibiting activity of the phosphonic acid derivatives of thepresent invention is shown in the following table. The inhibitingactivity is indicated by the concentration of inhibitors required toprovide 50% inhibition based on the amount converted to endothelin I inthe control plot, namely the 50% inhibition concentration (IC50).

    ______________________________________                                        Inhibiting Activity of Phosphonic Acid Derivatives of the                     Present Invention on Endothelin-Converting Enzyme                             Compound                  IC50 (μM)                                        ______________________________________                                        N-(Phenethylphosphonyl)-Leu--Trp.2Na                                                                    0.2                                                 N-(Isoamylphosphonyl)-Leu--Trp.2Na                                                                      0.7                                                 N-(Cyclohexylmethylphosphonyl)-Leu--Trp.2Na                                                             0.4                                                 N-(Phenethylphosphonyl)-Phe--Trp.Na                                                                     0.5                                                 N-(Phenethylphosphonyl)-Ile--Trp.2Na                                                                    1.2                                                 N-(Phenethylphosphonyl)-Val--Trp.2Na                                                                    2.8                                                 N-(Phenethylphosphonyl)-Cha--Trp.2Na                                                                    1.0                                                 N-(Phenethylphosphonyl)-Nle--Trp.2Na                                                                    1.4                                                 N-(Phenethylphosphonyl)-Leu--Phe.2Na                                                                    5.7                                                 N-(Phenethylphosphonyl)-Leu--Leu.2Na                                                                    4.8                                                 N-(1-Naphthylmethylphosphonyl)-Leu--Trp.2Na                                                             0.2                                                 N-(2-Naphthylmethylphosphonyl)-Leu--Trp.2Na                                                             1.0                                                 N- 2-(1-Naphthyl)ethylphosphonyl)-Leu--Trp.2Na                                                          0.4                                                 N- 2-(2-Naphthyl)ethylphosphonyl)-Leu--Trp.2Na                                                          0.1                                                 ______________________________________                                    

REFERENCE EXAMPLE

1. Preparation of Endothelin-Converting Enzyme Sample for AssayingEnzyme Inhibiting Activity

As endothelin-converting enzymes existing in organisms, two kinds ofmetalloenzymes different in enzymological properties are generallyknown. One exits in the cytoplasms, and the other is membrane-bound. Thelatter is characterized by that it is inhibited with phosphoramidonknown as an inhibitor for metalloproteases Matsumura et al., FEBS Lett.,272, 166-170 (1990)!. The endothelin-converting enzyme in the presentinvention means the membrane-bound enzyme of these two kinds of enzymes.

One example of the preparation thereof is hereinafter described.

About 500 g of the pig lung (of one pig) was sliced and disrupted in 2.0liter of PBS supplemented with 0.05% sodium azide, 1.0 mMdithiothreitol, 1.0 mM PMSF, 1.0 μg/ml leupeptin, 1.0 μg/ml chymostatin,1.0 μg/ml pepstatin A and 1.0 μM E-64, and then homogenized (1 minute×5times) by a Polytron mixer (KINEMATICA). The centrifuged supernatant(240×G, 5 minutes) was further centrifuged at 10,000×G for 15 minutes,and the resulting supernatant was subjected to ultracentrifuge(100,000×G, 90 minutes) to obtain membrane fraction as a precipitate. Tothis precipitate, 1.0% Triton X-100 was added to solubilize enzymeactivity. The protein amount of the solubilized membrane fractions wasabout 2.3 g. This was diluted to 2.0 mg of protein/ml to use forassaying the activity of enzyme inhibitors.

2. Measurement of Molecular Weight of Endothelin-Converting Enzyme

2.0 ml of an enzyme solution obtained by treatment similar to thatdescribed above using 1.0% 3-(3-cholamidopropyldimethylammonio!-1-propanesulfonic acid (CHAPS) inplace of 1.0% Triton X-100 as the solubilizing agent was subjected togel filtration using a HiLoad 16/60 Superdex 200 pg column (Pharmacia,1.6×600 mm) pre-equilibrated with 50 mM Trihydrochloric acid buffer (pH8.0) supplemented with 0.1% CHAPS, 0.15M sodium chloride, 0.02% sodiumazide, 0.1 mM PMSF, 1.0 μg/ml leupeptin, 1.0 μg/ml pepstatin A and 1.0μM E-64. The same buffer as used for equilibration was used as anelution, and the flow rate was 0.5 ml/minute. Separately taking 5.0 mlof each eluate, the endothelin-converting enzyme activity of eachfraction was assayed. The molecular weight of this enzyme was estimatedto be about 300,000 from a comparison between the elution position ofthe enzyme activity and that of the standard position for calibration ofmolecular weight analyzed under the same conditions. Chromatography wasconducted using a BioPilot system (Pharmacia).

3. Various Properties of Endothelin-Converting Enzyme

3-1. Optimum Ph

Using bis-Tris-hydrochloric acid buffer, Tris-hydrochloric acid bufferor diethanolamine-hydrochloric acid buffer, the enzyme activity wasassayed at various pHs. As a result, this enzyme has an optimum pHaround pH 7.5

3-2. Behavior to Various Protease Inhibitors

The inhibiting ratio of various protease inhibitors to this enzyme isshown in the following table. This enzyme was not inhibited so much be aspecific enzyme inhibitor to each of serine protease, SH protease andacid protease, and was inhibited by o-phenanthroline orethylenediaminetetraacetic acid which is a metal chelating agentinhibiting the activity of metalloprotease, and by phosphoramidone knownas a metalloprotease inhibitor. From the above, this enzyme wasconcluded to be a metalloenzyme. This enzyme was also inhibited by an SHreagent. A similar phenomenon is known to be observed in some kind ofmetalloprotease.

    ______________________________________                                        Inhibition Effect of Various Protease Inhibitors on                           Endothelin-Converting Enzyme                                                                     Concentration                                                                            Inhibiting                                      Inhibitor          (μM)    Ratio (%)                                       ______________________________________                                        Serine protease inhibitor                                                     PMSF               1000       32                                              Leupeptin          100        23                                              Chymostatin        100        2                                               SH protease inhibitor E-64                                                                       100        9                                               SH reagent Dithiothreitol                                                                        1000       96                                              Acid protease inhibitor                                                                          100        20                                              Pepstatin                                                                     Metalloprotease inhibitor                                                     o-Phenanthroline   1000       92                                              Ethylenediaminetetraacetic acid                                                                  1000       70                                              Phosphoramidone    100        70                                              ______________________________________                                    

3-3. Metal Requirement

Since this enzyme is a metalloenzyme, the metal requirement wasexamined. Various metals was added to the enzyme in the presence ofo-phenanthroline to examine the restoration of its activity. As aresult, this revealed that the activity of this enzyme was not restoredby addition of copper (divalence), but restored by addition of any metalions of manganese (divalence), zinc (divalence) and cobalt (divalence).This enzyme is therefore likely to contain any of these three kinds ofmetals at its active center.

3-4. Influence of Temperature

The activity of this enzyme at 37° C. is more than 3 times that at 25°C.

3-5. Kinetic Coefficient

The Michaelis coefficient of this enzyme to pig big endothelin wascalculated to be about 5×10⁻⁶ M from the Lineweaver-Burk plot.

EXAMPLE 1 N-(Phenethylphosphonyl)-Leu-Trp.2Na

(1) Boc-Leu-Trp-OBzl

Trp-OBzl.HCl (purchased from Kokusan Kagaku) (8.39 g) was dissolved inN,N-dimethylformamide (200 ml), and triethylamine (3.90 ml) andBoc-Leu-ONB prepared from Boc-Leu-OH.H₂ O (6.32 g), HONB (4.77 g) andDCC (5.49 g)! were added thereto under ice cooling, followed by stirringfor 12 hours. The solvent was removed by distillation under reducedpressure, and the residue was dissolved in ethyl acetate. The resultingsolution was washed with water, 10% aqueous citric acid, water,saturated aqueous sodium hydrogencarbonate and water in this order.After drying with anhydrous sodium sulfate, the solvent was removed bydistillation to obtain a crude product. Recrystallization from ethylacetate-petroleum ether gave needle crystalline colorlessBoc-Leu-Trp-OBzl (10.3 g).

Melting point: 131°-132° C., TLC Rf¹ 0.59

α!^(D) ₂₅ =-23.8° (C=1.04, MeOH)

elemental analysis: as C₂₉ H₃₇ N₃ O₅ Calculated: C: 68.62; H: 7.35; N:8.28. Found: C: 68.46; H: 7.53; N: 8.30.

(2) N-(O-Benzyl-P-Phenethylphosphonyl)-Leu-Trp-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml) wereadded to Boc-Leu-Trp-OBzl (1.20 g) obtained in (1) under ice cooling todissolve it, followed by stirring for 30 minutes. The solvent wasremoved by distillation under reduced pressure, and diethyl ether wasadded to the residue to precipitate crystals. The crystals were filteredoff, and dried. The resulting product was suspended in dichloromethane,and triethylamine (0.66 ml) and O-benzyl-P-phenethyl phosphochloridateprepared from dibenzylphenethyl phosphonate (868 mg) and phosphoruspentachloride (544 mg) by the method described in E. D. Thorsett et al.,Proc. Natl. Acad. Sci. USA, 79, 2176 (1982)! were added thereto underice cooling, followed by stirring for 12 hours. Dichloromethane (50 ml)was added to the reaction solution for dilution, and then water wasadded thereto. After stirring for 10 minutes, the product was extractedwith dichloromethane. The resulting dichloromethane solution was washedwith 10% aqueous citric acid, water, saturated aqueous sodiumhydrogencarbonate and water in this order. After drying with anhydroussodium sulfate, the solvent was removed by distillation. The residue waspurified by silica gel column chromatography. A crude product wasobtained from fractions eluted with chloroform. Recrystallization fromethyl acetate-petroleum ether gave needle crystalline colorlessN-(O-benzyl-P-phenethylphosphonyl)-Leu-Trp-bBzl (580 mg).

Melting point: 85°-87° C., TLC Rf¹ 0.31

Elemental analysis: as C₃₉ H₄₄ N₃ O₅ P Calculated: C: 70.36; H: 6.66; N:6.31. Found: C: 70.10; H: 6.64; N: 6.23.

IR νmax(KBr)cm⁻¹ : 1740 (C═O), 1660 (NHC═O), 1560 (Ar), 1190 (P═O)

NMR δppm(CDCl₃): 0.81-0.88 (6H, m), 1.27-1.37 (1H, m), 1.50-1.56 (1H,m), 1.60-1.70 (1H, m), 1.78-1.91 (1H, m), 1.93-2.00 (1H, m), 2.72-2.93(3H, m), 3.25-3.30 (2H, m), 3.73-3.88 (1H, m), 4.71-5.10 (5H, m),6.75-7.90 (22H, m)

(3) N-(Phenethylphosphonyl)-Leu-Trp.2Na

N-(O-Benzyl-P-phenethylphosphonyl-Leu-Trp-OBzl (70.0 mg) obtained in (2)and sodium hydrogencarbonate (7.7 mg) were dissolved in methanol-water(10:1) (11 ml), and 10% palladium-carbon (20 mg) was added thereto toconduct catalytic reduction in a stream of hydrogen at ordinarytemperature at ordinary pressure for 1.5 hours. The catalyst was removedby filtration, and the filtrate was concentrated under reduced pressure,followed by addition of water. Lyophilization gave powderyN-(phenethylphosphonyl)-Leu-Trp.2Na (40.3 mg).

TLC Rf¹ 0.58

LSIMS: m/z=530.2 M+H⁺ !

EXAMPLE 2 N-(Phenathylphosphonyl)-Ile-Trp.2Na

(1) Boc-Ile-Trp-OBzl

Trp-OBzl.HCI (purchased from Kokusan Kagaku) (8.39 g) was condensed withBOC-Ile-ONB prepared from Boc-Ile4H.0.5H₂ O (6.09 g), HONB (4.77 g) andDCC (5.49 g)! in a manner similar to that of Example 1-(1) to obtainneedle crystalline colorless Boc-Ile-Trp-OBzl (8.02 g).

Melting point: 111°-112° C., TLC Rf¹ 0.56

α!^(D) ₂₅ =-25.5° C. (C=1.05, MeOH)

Elemental analysis: as C₂₉ H₃₇ N₃ O₅ Calculated: C: 68.62; H: 7.35; N:8.28. Found: C: 68.50; H: 7.57; N: 8.23.

(2) N-(O-Benzyl-P-Phenethylphosphonyl)-Ile-Trp-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml) wereadded to Boc-Ile-Trp-OBzl (1.62 g) obtained in (1) under ice cooling todissolve it, followed by stirring for 30 minutes. The solvent wasremoved by distillation under reduced pressure, and diethyl ether wasadded to the residue to precipitate crystals. The crystals were filteredoff, and dried. The resulting product was reacted withO-benzyl-P-phenethyl phosphochloridate prepared from dibenzylphenethylphosphonate (1.17 g) and phosphorus pentachloride (800 mg)! in a mannersimilar to that of Example 1-(2) to obtain needle crystalline colorlessN-(O-benzyl-P-phenethylphosphonyl)-Ile-Trp-OBzl (390 mg).

Melting point: 151°-152° C., TLC Rf¹ 0.23

Elemental analysis: as C₃₉ H₄₄ N₃ O₅ P Calculated: C: 70.36; H: 6.66; N:6.31. Found: C: 70.10; H: 6.64; N: 6.23.

IR νmax(KBr)cm⁻¹ : 1730 (C═O), 1660 (NHC═O), 1540 (Ar), 1200 (P═O)

NMR δppm(CDCl₃): 0.70-0.88 (6H, m), 0.93-1.03 (1H, m), 1.33-1.42 (1H,m), 1.63-1.76 (1H, m), 1.79-1.93 (1H, m), 1.93-2.02 (1H, m), 2.55-2.91(2H, m), 2.94-3.02 (1H, m), 3.19-3.22 (2H, m), 3.59-3.67 (1H, m),4.73-5.11 (5H, m), 6.51-7.87 (22H, m)

(3) N-(Phenethylphosphonyl)-Ile-Trp.2Na

N-(O-Bezel-P-phenethylphosphonyl)-Ile-Trp-OBzl (70.0 mg) obtained in (2)was subjected to catalytic reduction in a manner similar to that ofExample 1-(3) to obtain powdery N-(phenethyl-phosphonyl)-Ile-Trp.2Na(45.8 mg).

TLC Rf² 0.58

LSIMS: m/z=530.2 M+H⁺ !

EXAMPLE 3 N-(Phenethylphosphonyl)-Val-Trp.2Na

(1) Boc-Val-Trp-OBzl

Trp-OBzl. HCl (purchased from Kokusan Kagaku) (500 g) was condensed withBoc-Val-ONB prepared from Boc-Val-OH (3.28 g), HONB (2.84 g) and DCC(3.27 g)! in a manner similar to that of Example 1-(1) to obtain needlecrystalline colorless Boc-Val-Trp-OBzl (4.88 g).

Melting point: 139°-140° C., TLC Rf¹ 0.44

α!^(D) ₂₅ =-21.8° (C=1.04, MeOH)

Elemental analysis: as C₂₈ H₃₅ N₃ O₅ Calculated: C: 68.13; H: 7.15; N:8.51.Found: C: 68.13; H: 7.08; N: 8.51.

(2) N-(O-Benzyl-P-Pbenethylphosphonyl).Val-Trp-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml) wereadded to Boc-Val-Trp-OBzl (1.58 g) obtained in (1) under ice cooling todissolve it, followed by stirring for 30 minutes. The solvent wasremoved by distillation under reduced pressure, and diethyl ether wasadded to the residue to precipitate crystals. The crystals were filteredoff and dried. The resulting product was reacted withO-benzyl-P-phenethyl phosphochloridate prepared from dibenzylphenethylphosphonate (1.17 g) and phosphorus pentachloride (800 mg)! in a mannersimilar to that of Example 1-(2) to obtain needle crystalline colorlessN-(O-benzyl-P-phenethylphosphonyl)-Val-Trp-OBzl (337 mg).

Melting point: 143°-144° C., TLC Rf¹ 0.23

Elemental analysis: as C₃₈ H₄₂ N₃ O₅ P.0.25H₂ O Calculated: C: 69.55; H:6.52; N: 6.40 Found: C: 69.53; H: 6.78; N: 6.31

IR μmax(KBr)cm⁻¹ : 1740 (C═O), 1660 (NHC═O), 1550 (Ar), 1200 (P═O)

NMR δppm(CDCl₃): 0.78-0.83 (3H, m), 0.87-0.92 (3H, m), 1.83-2.06 (3H,m), 2.76-2.93 (2H, m), 2.99-3.10 (1H, m), 3.19-3.32 (2H, m), 3.56-3.66(1H, m), 4.22-5.60 (SH, m), 6.55-7.91 (22H, m)

(3) N-(Phenethylphosphonyl)-Val-Trp.2Na

N-(O-Benzyl-P-phenethylphosphonyl)-Val-Trp-OBzl (70.0 mg) obtained in(2) was subjected to catalytic reduction in a manner similar to that ofExample 1-(3) to obtain powdery N-(phenethylphosphonyl)-Val-Trp.2Na(40.3 mg).

TLC Rf² 0.58

LSIMS: m/z=516.1 M+H⁺ !

EXAMPLE 4 N-(Phenethylphosphonyl)-Phe-Trp.2Na

(1) Boc-Phe-Trp-OBzl

Trp-OBzl.HCl (purchased from Kokusan Kagaku) (5.00 g) was condensed withBoc.-Phe-ONB prepared from Boc-Phe-OH (4.01 g), HONB (2.84 g) and DCC(3.27 g)! in a manner similar to that of Example 1-(1) to obtain needlecrystalline colorless Boc-Phe-Trp-OBzl (7.98 g).

Melting point: 129°-130° C., TLC Rf¹ 0.48

α!^(D) ₂₅ =-4.1° (C=1.04, MeOH)

Elemental analysis: as C₃₂ H₃₅ N₃ O₅ Calculated: C: 70.96; H: 6.51; N:7.76. Found: C: 70.70; H: 6.72; N: 7.53.

(2) N-(O-Benzyl-P-Phenethylphosphonyl)-Phe-Trp-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml) wereadded to Boc-Phe-Trp-OBzl (1.73 g) obtained in (1) under ice cooling todissolve it, followed by stirring for 30 minutes. The solvent wasremoved by distillation under reduced pressure, and diethyl ether wasadded to the residue to precipitate crystals. The crystals were filteredoff and dried. The resulting product was reacted withO-benzyl-P-phenethyl phosphochloridate prepared from dibenzylphenethylphosphonate (1.17 g) and phosphorus pentachloride (800 mg)! in a mannersimilar to that of Example 1-(2) to obtain needle crystalline colorlessN-(O-benzyl-P-phenethylphosphonyl)-Phe-TrP-OBzl (452 mg).

Melting point: 54°-55° C. TLC Rf¹ 0.30

Elemental analysis: as C₄₂ H₄₂ N₃ O₅ P Calculated: C: 72.09; H: 6.05; N:6.00. Found: C: 69.71; H: 6.42; N: 5.75.

IR νmax(KBr)cm⁻¹ : 1740 (C═O), 1660 (NHC═O), 1500 (Ar), 1200 (P═O)

NMR δppm(CDCl₃): 1.45-1.68 (2H, m), 2.50-2.66 (2H, m), 2.72-3.03 (3H,m), 3.12-3.30 (2H, m), 4.01-4.15 (1H, m), 4.36-4.64 (1H, m), 4.75-4.83(1H, m), 4.41-5.09 (3H, m), 6.60-7.87 (2H, m)

(3) N-(phenethylphosphonyl)-Phe-Trp.2Na

N-(O-Benzyl-p-phenethylphosphonyl)-Phe-TrP-OBzl (70 mg) obtained in (2)was subjected to catalytic reduction in a manner similar to that ofExample 1-(3) to obtain powdery N-(phenethylphosphonyl) -Phe-Trp.2Na(45.0 mg).

TLC Rf² 0.58

LSIMS: m/z=564.1 M+H⁺ !

EXAMPLE 5 N-(phenethylphosphonyl)-Cha-Trp.2Na

(1) Boc-Cha-Trp-OBzl

Trp-OBzl.HCl (purchased from Kokusan Kagaku) (1.65 g) was condensed withBoc-Cha-ONB prepared from Boc-Cha-OH.DCHA (purchased from Nova Biochem)(2.26 g), HONB (941 mg) and DCC (1.08 g)! in a manner similar to that ofExample 1-(1) to obtain needle crystalline colorless Boc-Cha-Trp-OBzl(2.12 g).

Melting point: 67°-68° C., TLC Rf¹ 0.70

α!^(D) ₂₅ =-14.9° (C=1.02, MeOH)

Elemental analysis: as C₃₂ H₄₁ N₃ O₅ Calculated: C: 70.18; H: 7.55; N:7.67. Found: C: 70.23; H: 7.84; N: 7.38.

(2) N-(O-Benzyl-P-Phenethylphosphonyl)-Cha-TrP-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml) wereadded to Boc-Cha-Trp-OBzl (1.75 g) obtained in (1) under ice cooling todissolve it, followed by stirring for 30 minutes. The solvent wasremoved by distillation under reduced pressure, and diethyl ether wasadded to the residue to precipitate crystals. The crystals were filteredoff and dried. The resulting product was reacted withO-benzyl-P-phenethyl phosphochloridate prepared from dibenzylphenethylphosphonate (1.17 g) and phosphorus pentachloride (800 mg)! in a mannersimilar to that of Example 1-(2) to obtain needle crystalline colorlessN-(O-benzyl-p-phenethylphosphonyl)-Cha-Trp-OBzl (41o mg).

Melting point: 107°-109° C., TLC Rf¹ 0.28

Elemental analysis: as C₄₂ H₄₈ N₃ O₅ P Calculated: C: 71.47; H: 6.85; N:5.95. Found: C: 71.38; H: 6.93; N: 5.58.

IR νmax(KBr)cm⁻¹ : 1750 (C═O), 1660 (NHC═O), 1500 (Ar), 1200 (P═O).

NMR δppm(CDCl₃): 0.76-0.93 (2H, m), 1.04-1.21 (3H, m), 1.26-1.41 (2H,m), 1.53-1.73 (6H, m), 1.84-2.01 (2H, m), 2.71-2.91 (3H, m), 3.24-3.31(2H, m), 3.78-3.87 (1H, m), 4.70-5.11 (5H, m), 6.71-7.96 (22H, m)

(3) N-(Phenethylphosphonyl)-Cha-Trp.2Na

N-(O-Benzyl-P-phenethylphosphonyl)-Cha-Trp-OBzl (70.6 mg) obtained in(2) was subjected to catalytic reduction in a manner similar to that ofExample 1-(3) to obtain powdery N-(phenethylphosphonyl)-Val-Trp.2Na(38.4 mg).

TLC Rf² 0.58

LSIMS: m/z=570.2 M+H⁺ !

EXAMPLE 6 N- phenethylphosphonyl)-Nle-Trp.2Na

(1) Boc-Nle-Trp-OBzl

Trp-OBzl.HCI (purchased from Kokusan Kagaku) (1.65 g) was condensed withBoc-Nle-ONB prepared from Boc-Nle-OH.DCHA (purchased from Nova Biochem)(2.06 g), HONB (941 mg) and DCC (1.08 g)! in a manner similar to that ofExample 1-(1) to obtain oily light yellow Boc-Nle-Trp.OBzl (2.41 g).

TLC R^(f) 0.55

LSIMS: m/z=508.2 (M⁺)

IR νmax(KBr)cm⁻¹ : 1680 (NHC═O), 1510 (Ar)

NMR δppm(CDCl₃): 0.83 (3H, t), 1.18-1.31 (4H, m), 1.41 (9H, s),1.65-1.80 (2H, m), 3.25-3.35 (2H,m), 4.04 (1H, broad,s), 4.40-4.99 (2H,m), 5.07 (2H, s), 6.50-6.55 (1H, m), 6.94-7.51 (10H,m), 8.13 (1H, broad,s)

(2) N-(O-Benzyl-P-Phenethylphosphonyl)-Nle-Trp-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml) wereadded to Boc-Nle.Trp.OBzl (1.62 g) obtained in (1) under ice cooling todissolve it, followed by stirring for 30 minutes. The solvent wasremoved by distillation under reduced pressure, and diethyl ether wasadded to the residue to precipitate crystals. The crystals were filteredoff and dried. The resulting product was reacted withO-banzyl-P-phenethyl phosphochloridate prepared from dibenzylphenethylphosphonate (1.17 g) and phosphorus pentachloride (800 mg)! in a mannersimilar to that of Example 1-(2) to obtain oily colorlessN-(O-benzyl-P-phenethylphosphonyl)-Nle-Trp-OBzl (150 mg).

TLC Rf¹ 0.27

LSIMS: m/z=666.3 (M⁺)

IR νmax(KBr)cm⁻¹ : 1740 (C═O), 1660 (NHC═O), 1500 (Ar), 1190 (P═O)

NMR δppm(CDCl₃): 0.80-0.87 (3H, m), 1.16-1.28 (4H, m), 1.41-1.83 (2H,m), 1.83-2.01 (2H, m), 2.74-2.90 (3H, m), 3.25-3.30 (2H, m), 3.67-3.79(1H, m), 4.73-5.13 (5H, m), 6.66-7.85 (22H, m)

(3) N-(Phenethylphosphonyl)-Nle-Trp.2Na

N-(O-Benzyl-P-phenethylphosphonyl)-Nle-Trp-OBzl (31.0 mg) obtained in(2) was subjected to catalytic reduction in a manner similar to that ofExample 1-(3) to obtain powdery N- phenethylphosphonyl)-Nle-Trp.2Na(13.3 mg).

TLC Rf² 0.58

LSIMS: m/z=530.2 M+H⁺ !

EXAMPLE 7 N-(Phenethylphosphonyl)-Leu-Phe.2Na

(1) Boc-Leu-Phe-OBzl

Phe-OBzl.Tos (purchased from Peptide Laboratory) (7.27 g) was condensedwith Boc-Leu-ONB prepared from Boc-Leu-OH.H₂ O (3.93 g), HONB (3.20 mg)and DCC (3.68 8)! in a manner similar to that of Example 1-(1) to obtainneedle crystalline colorless Boc-Leu-Phe-OBzl (5.64 g).

Melting point: 100°-101° C., TLC Rf¹ 0.66

α!^(D) ₂₅ =-35.3° (C=1.04, MeOH)

Elemental analysis: as C₂₇ H₃₆ N₂ O₅ Calculated: C: 69.21; H: 7.74; N:5.98. Found: C: 69.42; H: 7.70; N: 6.05.

(2) N-(O-Benzyl-P-Phenethylphosphonyl)-Leu-phe-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml) wereadded to Boc-Leu-Phe-OBzl (1.50 g) obtained in (1) under ice cooling todissolve it, followed by stirring for 30 minutes. The solvent wasremoved by distillation under reduced pressure, and diethyl ether wasadded to the residue to precipitate crystals. The crystals were filteredoff and dried. The resulting product was reacted withO-benzyl-P-phenethyl phosphochloridate prepared from dibenzylphenethylphosphonate (1.17 g) and phosphorus pentachloride (800 mg)! in a mannersimilar to that of Example 1-(2) to obtain needle crystalline colorlessN-(O-benzyl-P-phenethylphosphonyl)-Leu-Phe-OBzl (351 mg).

Melting point: 93°-95° C. TLC Rf¹ 0.37

Elemental analysis: as C₃₇ H₄₃ N₂ O₅ P Calculated: C: 70.91; H: 6.92; N:4.47. Found: C: 70.64; H: 7.04; N: 4.30.

IR νmax(KBr)cm⁻¹ : 1750 (C═O), 1660 (NHC═O), 1500 (Ar), 1190 (P═O)

NMR δppm(CDCl₃): 0.85-0.90 (6H, m), 1.23-1.37 (1H, m), 1.48-1.56 (1H,m), 1.59-1.69 (1H, m), 1.92-2.06 (1H, m), 2.26-2.95 (3H, m), 3.01-3.13(2H, m), 3.73-3.81 (1H, m), 4.87-5.16 (5H, m), 6.69-7.37 (21H, m)

(3) N-(Phenethylphosphonyl)-Leu-Phe.2Na

N-(O-Benzyl-P-phenethylphosphonyl)-Leu-Phe-OBzl (70.0 mg) obtained in(2) as subjected to catalytic reduction in a manner similar to that ofExample 1-(3) to obtain powdery N-(phenethylphosphonyl)-Leu-Phe.2Na(52.4 mg).

TLC Rf² 0.58

LSIMS: m/z=491.1 M+H⁺ !

EXAMPLE 8 N-(Phenethylphosphonyl)-Leu-Leu.2Na

(1) Boc-Leu-Leu-OBzl

Leu-OBzl.Tos (purchased from Peptide Laboratory) (6.69 g) was condensedwith Boc-Leu-ONB prepared from Boc-Leu-OH.H₂ O (3.93 g), HONB (3.20 mg)and DCC (3.68 g)! in a manner similar to that of Example 1-(1) to obtainneedle crystalline colorless Boc-Leu-Leu-OBzl (5.67 g).

Melting point: 90°-91° C., TLC Rf¹ 0.94

α!^(D) ₂₅ =-51.0° (C=1.00, MeOH)

Elemental analysis: as C₂₄ H₃₈ N₂ O₅ Calculated: C: 66.33; H: 8.81; N:6.45. Found: C: 66.29; H: 8.78; N: 6.48.

(2) N-O-Benzyl-P-Phenethylphosphonyl)-Leu-Leu-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml) wereadded to Boc-Leu-Leu-OBzl (1.39 g) obtained in (1) under ice cooling todissolve it, followed by stirring for 30 minutes. The solvent wasremoved by distillation under reduced pressure, and diethyl ether wasadded to the residue to precipitate crystals. The crystals were filteredoff and dried. The resulting product was reacted withO-benzyl-P-phenethyl phosphochloridate prepared from dibenzylphenethylphosphonate (1.17 g) and phosphorus pentachloride (800 mg)! in a mannersimilar to that of Example 1-(2) to obtain needle crystalline colorlessN(O-benzyl-P-phenethylphosphonyl)-Leu-Leu-OBzl (385 mg).

Melting point: 141°-143° C., TLC Rf¹ 0.52

Elemental analysis: as C₃₄ H₄₅ N₂ O₅ P Calculated: C: 68.90; H: 7.65; N:4.73. Found: C: 68.64; H: 7.76; N: 4.50.

IR νmax(KBr)cm⁻¹ : 1750 (C═O), 1660 (NHC═O), 1500 (Ar), 1200 (P═O)

NMR δppm(CDCl₃): 0.81-0.95 (12H, m), 1.36-1.76 (6H,m), 2.00-2.14 (2H,m), 2.83-3.14 (3H, m), 4.80-4.90 (1H, m), 4.60-4.66 (1H, m), 4.86-5.16(4H, m), 6.75-7.39 (16H, m)

(3) N-(Phenethylphosphonyl)-Leu-Leu.2Na

N-(O-Benzyl-P-phenethylphosphonyl)-Leu-Leu-OBzl (70.0 mg) obtained in(2) was subjected to catalytic reduction in a manner similar to that ofExample 1-(3) to obtain powdery N-(phenethylphosphonyl)-Leu-Leu.2Na(51.0 mg).

TLC Rf² 0.58

LSIMS: m/z=457.1 M+H⁺ !

EXAMPLE 9 N-(Isoamylphosphonyl)-Leu-Trp.2Na

(1) N-(O-Benzyl-P-Isoamylphosphonyl)-Leu-Trp-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml)-wereadded to Boc-Leu-Trp-OBzl (1.20 g) obtained in Example I-(1) under icecooling to dissolve it, followed by stirring for 30 minutes. The solventwas removed by distillation under reduced pressure, and diethyl etherwas added to the residue to precipitate crystals. The crystals werefiltered off and dried. The resulting product was reacted withO-benzyl-P-isoamyl phosphochloridate prepared from dibenzylisoamylphosphonate (788 mg) and phosphorus pentachloride (544 mg) by the methoddescribed in E. D. Thorsett et al., Proc. Natl. Acad. Sci. USA. 79, 2176(1982)! in a manner similar to that of Example 1-(2) to obtain needlecrystalline colorless N-(O-benzyl-P-isoamylphosphonyl)-Leu-Trp-OBzl (253mg).

Melting point: 78°-80° C., TLC Rf¹ 0.30

Elemental analysis: as C₃₆ H₄₆ N₃ O₅ P Calculated: C: 68.44; H: 7.34; N:6.65. Found: C: 68.23; H: 7.10; N: 6.61.

IR νmax(KBr)cm⁻¹ : 1730 (C═O), 1660 (NHC═O), 1530 (Ar), 1200 (P═O)

NMR δppm(CDCl₃): 0.80-0.87 (12H, m), 1.31-1.70 (8H,m), 2.73-2.86 (1H,m), 3.23-3.35 (2H, m), 3.71-3.82 (1H, m), 4.65-5.11 (5H, m), 6.76-8.13(17H, m)

(2) N-(Isoamylphosphonyl)-Leu-Trp.2Na

N-(O-Benzy)-P-isoamylphosphonyl)-Leu-Trp-OBzl (70.0 mg) obtained in (1)was subjected to catalytic reduction in a manner similar to that ofExample 1-(3) to obtain powdery N-(isoamylphosphonyl)-Leu-Trp.2Na (54.0mg).

TLC Rf² 0.58

LSIMS: m/z=496.1 M+H⁺ !

EXAMPLE 10 N-(Cyclohexylmethylphosphonyl)-Leu-Trp.2Na

(1) N-(O-Benzyl-P-Cyclohexylmethylphosphonyl)-Leu-Trp-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml) wereadded to Boc-Leu-Trp-OBzl (1.20 g) obtained in Example 1-(1) under icecooling to dissolve it, followed by stirring for 30 minutes. The solventwas removed by distillation under reduced pressure, and diethyl etherwas added to the residue to precipitate crystals. The crystals werefiltered off and dried. The resulting product was reacted withO-benzyl-P-cyclohexylmethyl phosphochloridate prepared fromdibenzylcyclohexylmethyl phosphonate (1.10 g) and phosphoruspentachloride (767 mg) by the method described in E. D. Thorsett et al.,Proc. Natl. Acad. Sci. USA, 79, 2176 (1982)! in a manner similar to thatof Example 1-(2) to obtain needle crystalline colorlessN-(O-benzyl-P-cyclohexylmethylphosphonyl)-Leu-Trp-OBzl (360 mg).

Melting point: 99°-101° C., TLC Rf¹ 0.32

Elemental analysis: as C₃₈ H₄₈ N₃ O₅ P Calculated: C: 69.39; H: 7.36; N:6.39. Found: C: 69.13; H: 7.42; N: 6.31.

IR νmax(KBr)cm⁻¹ : 1740 (C═O), 1660 (NHC═O), 1560 (Ar), 1200 (P═O)

NMR δppm(CDCl₃): 0.81-0.88 (6H, m), 0.88-1.99 (16H,m), 2.92-3.05 (1H,m), 3.22-3.35 (2H, m), 3.70-3.80 (1H, m), 4.66-5.10 (SH, m), 6.35-8.31(17H, m)

(2) N-(Cyclohexylmetylphosphonyl)-Leu-Trp.2Na

N-(O-Benzyl-P-cyclohexylmethylphosphonyl)-Leu-TrP-OBzl (70.0 mg)obtained in (1) was subjected to catalytic reduction in a manner similarto that of Example 1-(3) to obtain powderyN-(cyclohexylmethylphosphonyl)-Leu-TrP.2Na (48.1 mg).

TLC Rf² 0.58

LSIMS: m/z=522.2 M+H^(+!)

EXAMPLE 11 N-(1-Naphthylmethylphosphonyl)-Leu-TrP.2Na

(1) N- O-Benzyl-P-(1-Naphthyl)methylphosphonyl!-Leu-TrP-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml) wereadded to Boc-Leu-Trp-OBzl (1.64 g) obtained in Example 1-(1) under icecooling to dissolve it, followed by stirring for 30 minutes. The solventwas removed by distillation under reduced pressure, and diethyl etherwas added to the residue to precipitate crystals. The crystals werefiltered off and dried. The resulting product was reacted withO-benzyl-P-(1-naphthyl)methyl phosphochloridate prepared from dibenzyl1-naphthylmethyl phosphonate (1.30 g) and phosphorus pentachloride (792mg) by the method described in E. D. Thorsett et al., Proc. Natl. Acad.Sci. USA, 79, 2176 (1982)! in a manner similar to that of Example 1-(2)to obtain needle crystalline colorless N-O-benzyl-P-1-naphthyl)methylphosphonyl!-Leu-Trp-OBzl (560 mg).

Melting point: 54.0°-56.0° C., TLC Rf¹ 0.42

Elemental analysis: as C₄₂ H₄₄ N₃ O₅ P Calculated: C: 71.88; H: 6.32; N:5.99. Found: C: 71.77; H: 6.45; N: 5.95.

IR νmax(KBr)cm⁻¹ : 1740 (C═O), 1660 (NHC═O), 1510 (Ar), 1210 (P═O)

NMR δppm(CDCl₃): 0.60-0.81 (6H, m), 0.92-1.00 (1H, m), 1.20-1.57 (2H,m), 2.65-2.80 (1H, m), 3.20-3.81 (5H, m), 4.61-5.10 (5H, m), 6.57-8.05(24H, m)

(2) N-(1-Naphthylmethylphosphonyl)-Leu-Trp.2Na

N- O-Benzy!-P-(1-naphthyl)methylphosphonyl!-Leu-Trp-OBzl (30.0 mg)obtained in (1) was subjected to catalytic reduction in a manner similarto that of Example 1-(3) to obtain powderyN-(1-naphthylmethylphosphonyl)-Leu-Trp.2Na (17.5 mg).

TLC Rf² 0.62

LSIMS: m/z=566.1 M+H⁺ !

EXAMPLE 12 N-(2-Naphthylmethylphosphonyl)-Leu-Trp.2Na

(1) N- O-Benzyl-P-(2-Naphthyl)methylphosphonyl!-Leu-Trp-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml) wereadded to Boc-Leu-Trp-OBzl (1.64 g) obtained in Example 1-(1) under icecooling to dissolve it, followed by stirring for 30 minutes. The solventwas removed by distillation under reduced pressure, and diethyl etherwas added to the residue to precipitate crystals. The crystals werefiltered off and dried . The resulting product was reacted withO-benzyl-P-(2-naphthyl)methyl phosphochloridate prepared from dibenzyl2-naphthylmethyl phosphonate (1.30 g) and phosphorus pentachloride (792mg) by the method described in E. D. Thorsett et al., Proc. Natl. Acad.Sci. USA, 79, 2176 (1982)! in a manner similar to that of Example 1-(2)to obtain needle crystalline colorless N-O-benzyl-P-(2-naphthyl)methylphosphonyl!-Leu-Trp-OBzl (160 mg).

Melting point: 59.0°-61.0° C., TLC Rf¹ 0.45

Elemental analysis: as C₄₂ H₄₄ N₃ O₅ P Calculated: C: 71.88; H: 6.32; N:5.99. Found: C: 71.59; H: 6.38; N: 5.97.

IR νmax(KBr)cm⁻¹ : 1740 (C═O), 1660 (NHC═O), 1460 (Ar), 1220 (P═O)

NMR δppm(CDCl₃): 0.63-0.81 (6H, m), 1.10-1.29 (1H, m), 1.40-1.50 (1H,m), 1.50-1.63 (1H, m), 2.64-2.76 (1H, m), 3.02-3.30 (4H, m), 3.69-3.80(1H, m), 4.65-5.10 (5H, m), 6.48-7.97 (24H, m)

(2) N-(2-Naphthylmethylphosphonyl)-Leu-Trp.2Na

N- O-Benzyl-P-(2-naphthyl)methylphosphonyl!-Leu-Trp-OBzl (50.0 mg)obtained in (1) was subjected to catalytic reduction in a manner similarto that of Example 1-(3) to obtain powderyN-(2-naphthylmethylphosphonyl)-Leu-Trp.2Na (24.5 mg).

TLC Rf² 0.65

LSIMS: m/z=566.1 M+H⁺ !

EXAMPLE 13 N- 2-(1-Naphthyl)ethylphosphonyl!-Leu-Trp.2Na

(1) N- O-Benzyl-P- 2-(1-Naphthyl)!ethylphosphonyl!-Leu-Trp-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml) wereadded to Boc-Leu-Trp-OBzl (2.17 g) obtained in Example 1-(1) under icecooling to dissolve it, followed by stirring for 30 minutes. The solventwas removed by distillation under reduced pressure, and diethyl etherwas added to the residue to precipitate crystals. The crystals werefiltered off and dried. The resulting product was reacted withO-benzyl-P- 2-(1-naphthyl)!ethyl phosphochloridate prepared fromdibenzyl 2-(1-naphthyl)ethyl phosphonate (1.65 g) and phosphoruspentachloride (1.07 g) by the method described in E. D. Thorsett et al.,Proc. Natl. Acad. Sci. USA, 79, 2176 (1982)! in a manner similar to thatof Example 1-(2) to obtain needle crystalline colorless N- O-benzyl-P-2-(1-naphthyl)!ethylphosphonyl!-Leu-Trp-Bzl (420 mg).

Melting point: 51.5°-54.0° C., TLC Rf¹ 0.28

Elemental analysis: as C₄₃ H₄₆ N₃ O₅ P Calculated: C: 72.15; H: 6.48; N:5.87. Found: C: 72.08; H: 6.40; N: 5.74.

IR νmax(KBr)cm⁻¹ : 1740 (C═O), 1660 (NHC═O), 1460 (Ar), 1200 (P═O)

NMR δppm(CDCl₃): 0.75-0.90 (6H, m), 1.23-1.39 (1H, m), 1.46-1.71 (2H,m), 1.83-2.00 (1H, m), 2.00-2.13 (1H, m), 2.75-2.83 (1H, m), 3.13-3.38(4H, m), 3.75-3.87 (1H, m), 4.73-5.60 (5H, m), 6.66-7.97 (24H, m)

(2) N- 2-(1-Naphthyl)ethylphosphonyl!-Leu-Trp.2Na

N- O-Benzyl-P- 2-(1-naphthyl)ethylphosphonyl!-Leu-Trp-OBzl (50.0 mg)obtained in (1) was subjected to catalytic reduction in a manner similarto that of Example 1-(3) to obtain powderyN-12-(1-naphthyl)ethylphosphonyl!-Leu-Trp.2Na (31.2 mg).

TLC Rf² 0.63

LSIMS: m/z=580.1 M+H⁺ !

EXAMPLE 14 N- 2-(2-Naphthyl)ethylphosphonyl!-Leu-Trp.2Na

(1) N- O-Benzyl-P- 2-(2-Naphthyl)!ethylphosphonyl!-Leu-Trp-OBzl

1,2-Ethanedithiol (0.2 ml) and 8N hydrochloric acid-dioxane (20 ml) wereadded to Boc-Leu-Trp-OBzl (2.17 g) obtained in Example 1-(1) under icecooling to dissolve it, followed by stirring for 30 minutes. The solventwas removed by distillation under reduced pressure, and diethyl etherwas added to the residue to precipitate crystals. The crystals werefiltered off and dried. The resulting product was reacted withO-benzyl-P- 2-(2-naphthyl)!ethyl phosphochloridate prepared fromdibenzyl 2-(2-naphthyl)ethyl phosphonate (1.65 g) and phosphoruspentachloride (1.07 g) by the method described in E. D. Thorsett et al.,Proc. Natl. Acad. Sci. USA, 79, 2176 (1982)! in a manner similar to thatof Example 1-(2) to obtain needle crystalline colorless N- O-benzyl-P-2-(2-naphthyl)!ethylphosphonyl!-Leu-Trp-OBzl (850 mg).

Melting point: 51.5°-53.0° C., TLC Rf¹ 0.40

Elemental analysis: as C₄₃ H₄₆ N₃ O₅ P Calculated: C: 72.15; H: 6.48; N:5.87. Found: C: 72.05; H: 6.42; N: 5.79.

IR νmax(KBr)cm⁻¹ : 1740 (C═O), 1660 (NHC═O), 1510 (Ar), 1200 (P═O)

NMR δppm(CDCl₃): 0.80-0.88 (6H, m), 1.25-1.37 (1H, m), 1.50-1.56 (1H,m), 1.56-1.70 (1H, m), 1.91-2.00 (1H, m), 2.00-2.10 (1H, m), 2.68-2.81(1H, m), 2.89-3.06 (2H, m), 3.26-3.30 (2H, m), 3.73-3.84 (1H, m),4.72-5.10 (5H, m), 6.65-7.84 (24H, m)

(2) N- 2-(2-Naphthyl)ethylphosphonyl!-Leu-Trp.2Na

N- O-Benzyl-P- 2-(2-naphthyl)ethylphosphonyl!-Leu-Trp-OBzl (50.0 mg)obtained in (1) was subjected to catalytic reduction in a manner similarto that of Example 1-(3) to obtain powdery N-2-(2-naphthyl)ethylphosphonyl!-Leu-Trp.2Na (26.1 mg).

TLC Rf² 0.66

LSIMS: m/z=580.1 M+H⁺ !

What is claimed is:
 1. A compound represented by formula I! or apharmaceutically acceptable salt thereof: ##STR6## wherein R₁ is C₁₋₁₂alkyl group which may be substituted by C₃₋₈ cycloalkyl, halogen hydroxywhich may be protected, C₁₋₂ alkoxy, ketone or amino which may beprotected, (ii) a 5 to 7 member cycloalkyl group which may besubstituted by lower alkyl having 1 to 3 carbon atoms, halogen, hydroxywhich may be protected, C₁₋₂ alkoxy, keto, or amino which may beprotected or (iii) an aralkyl group which may be substituted by loweralkyl having 1 to 3 carbon atoms, C₅₋₆ cycloalkyl, halogen, hydroxywhich may be protected, or C₁₋₂ alkoxy;R₂ is (i) a C₁₋₈ alkyl group (ii)a cyclohexylmethylene group or (iii) a benzyl group; and R₃ is . .anindolylmethyl group or.!. a benzyl group.Iadd., except compounds inwhich (i) R₂ is a methyl group and (ii) R₁ isbenzyloxycarbonylaminomethyl, R₂ is isobutyl and R₃ is benzyl.Iaddend..2. The compound according to claim 1, in which R₁ is selected from thegroup which consists of an alkyl group having 1 to 12 carbon atoms; a5-, 6- or 7-membered alicyclic alkyl group; and an alkyl group having 1to 5 carbon atoms substituted by an aromatic hydrocarbon group having 6to 12 carbon atoms.
 3. The compound according to claim 2, in which saidalkyl group having 1 to 12 carbon atoms is isoamyl or cyclohexylmethyl,and said alkyl group having 1 to 5 carbon atoms substituted by thearomatic hydrocarbon group is phenylethyl naphthylmethyl ornaphthylethyl. . .4. The compound according to claim 1, in which saidindolylmethyl group is indol-3-ylmethyl..!.5. A compound represented byformula I! or a pharmaceutically acceptable salt thereof: ##STR7##wherein R₁ is isoamyl, cyclohexylmethyl, phenethyl, naphthylmethyl ornaphthylethyl R₂ is isobutyl or benzyl, and R₃ is indol-3-ylmethyl. 6.The compound according to claim 5, in which said compound isN-(phenethylphosphonyl)-leucyl-tryptophan.
 7. The compound according toclaim 5, in which said compound isN-(cyclohexylmethylphosphonyl)-leucyl-tryptophan.
 8. The compoundaccording to claim 5, in which said compound isN-(1-naphthylmethylphosphonyl)-leucyl-tryptophan.
 9. The compoundaccording to claim 5, in which said compound isN-{2-(1-naphthyl)ethylphosphonyl}-leu-cyl-tryptophan.
 10. Tne compoundaccording to claim 5, in which said compound isN-{2-(2-naphthyl)ethylphosphonyl}-leucyl-tryptophan.
 11. Apharmaceutical composition comprising the compound or thepharmaceutically acceptable salt thereof claimed in claim 5 and apharmaceutically acceptable carrier.
 2. A method for bringing aboutendothelin-converting enzyme inhibiting activity in a warm-blood animal,which comprises administering an effective amount of the compound or thepharmaceutically acceptable salt thereof claimed in claim 5 to thewarm-blood animal.
 13. A compound represented by formula I! or apharmaceutically acceptable sat thereof: ##STR8## wherein R₁ is (i) aC₁₋₁₂ alkyl group which may be substituted by C₃₋₈ cycloalkyl, halogen,hydroxy which may be protected, C₁₋₂ alkoxy, or amino which may beprotected, (ii) a 5 to 7 member cycloalkyl group which may besubstituted by lower alkyl having 1 to 3 carbon atoms, halogen, hydroxywhich may be protected, C₁₋₂ alkoxy, keto, or amino which may beprotected, or (iii) a C₇₋₁₇ aralkyl group which may be substituted bylower alkyl having 1 to 3 carbon atoms, C₅₋₆ cycloalkyl, halogen,hydroxy which may be protected, or C₁₋₂ alkoxy;R₂ is (i) a C₁₋₈ alkylgroup, (ii) a 5 to 7 member cycloalkyl group, or (iii) a benzyl group;and R₃ is a C₁₋₈ alkyl group which is substituted by 3-indolyl.Iadd.,except compounds in which R₂ is a methyl group.Iaddend..
 14. A compoundrepresented by formula I! or a pharmaceutically acceptable salt thereof:##STR9## wherein R₁ is phenethyl, isoamyl, cyclohexylmethylnaphthylmethyl or naphthylethyl, R2 is propyl, butyl, cyclohexylmethylor benzyl and R₃ is benzyl or indolylmethyl.
 15. A compound representedby formula I! or a pharmaceutically acceptable salt thereof: ##STR10##wherein R₁ is an alkyl group of 1 to 12 carbon atoms or an alkyl grouphaving 1 to 5 carbon atoms substituted by an aromatic hydrocarbon grouphaving 6 to 12 carbon atoms;R₂ is an alkyl group having 1 to 8 carbonatoms; an alkyl group having 1 to 8 carbon atoms substituted by acycloalkyl group having 5 to 7 carbon atoms; or an alkyl group having 1to 5 carbon atoms substituted by an aromatic hydrocarbon group having 6to 12 carbon atoms; and R₃ is alkyl having 1 to 8 carbon atomssubstituted by indolyl or an aromatic hydrocarbon group having . .to.!.6 to 12 carbon atoms.Iadd., except compounds in which R₂ is a methylgroup.Iaddend..
 16. The compound of claim 15 wherein R₁ is isoamyl,cyclohexylmethyl, phenylethyl, naphthylmethyl or naphthylethyl; R₂ isisobutyl or benzyl, and R₃ is indolylmethyl or benzyl. .Iadd.17. Thecompound according to claim 1, in which R₂ is n-propyl, isopropyl,isobutyl, sec-butyl, cyclohexylmethyl or benzyl. .Iaddend..Iadd.18. Amethod for bringing about endothelin-converting enzyme inhibitingactivity in a warm-blood animal, which comprises administering aneffective amount of the compound or the pharmaceutically acceptable saltthereof claimed in claim 1 to the warm-blood animal. .Iaddend..Iadd.19.A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and the compound of claim 1 or a pharmaceutically acceptablesalt thereof. .Iaddend..Iadd.20. The compound according to claim 13, inwhich R₂ is (i) a C₃₋₈ alkyl group, (ii) a 5 to 7 member cycloalkylgroup, or (iii) a benzyl group. .Iaddend..Iadd.21. The compoundaccording to claim 13, in which R₂ is n-propyl, isopropyl, isobutyl,sec-butyl, cyclohexylmethyl or benzyl. .Iaddend..Iadd.22. A method forbringing about endothelin-converting enzyme inhibiting activity in awarm-blooded animal, which comprises administering an effective amountof the compound or the pharmaceutically acceptable salt thereof claimedin claim 13 to the warm-blooded animal. .Iaddend..Iadd.23. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and the compound of claim 13 or a pharmaceutically acceptablesalt thereof. .Iaddend..Iadd.24. The compound according to claim 14, inwhich R₃ is indolylmethyl. .Iaddend..Iadd.25. The compound according toclaim 15, in which R₂ is an alkyl group having 3 to 8 carbon atoms; analkyl group having 1 to 8 carbon atoms substituted by a cycloalkyl grouphaving 5 to 7 carbon atoms; or an alkyl group having 1 to 5 carbon atomssubstituted by an aromatic hydrocarbon group having 6 to 12 carbonatoms; and R₃ is alkyl having 1 to 8 carbon atoms substituted byindolyl. .Iaddend..Iadd.26. The compound according to claim 15, in whichR₂ is n-propyl, isopropyl, isobutyl, sec-butyl, cyclohexylmethyl orbenzyl. .Iaddend..Iadd.27. A method for bringing aboutendothelin-converting enzyme inhibiting activity in a warm-bloodedanimal, which comprises administering an effective amount of thecompound or the pharmaceutically acceptable salt thereof claimed inclaim 15 to the warm-blooded animal. .Iaddend..Iadd.28. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and thecompound of claim 15 or a pharmaceutically acceptable salt thereof..Iaddend..Iadd.29. The compound of claim 16 wherein R₃ is indolylmethyl..Iaddend..Iadd.30. A compound represented by formula I! or apharmaceutically acceptable salt thereof: ##STR11##.Iaddend.wherein:R₁is (i) a C₁₋₁₂ alkyl group which may be substituted by C₃₋₈ cycloalkyl,halogen, hydroxy which may be protected, C₁₋₂ alkoxy, ketone or aminowhich may be protected, (ii) a 5 to 7 member cycloalkyl group which maybe substituted by lower alkyl having 1 to 3 carbon atoms, halogen,hydroxy which may be protected, C₁₋₂ alkoxy, keto, or amino which may beprotected or (iii) an aralkyl group which may be substituted by loweralkyl having 1 to 3 carbon atoms, C₅₋₆ cycloalkyl, halogen, hydroxywhich may be protected, or C₁₋₂ alkoxy; R₂ is (i) a C₁₋₈ alkyl group(ii) a cyclohexylmethylene group or (iii) benzyl group and R₃ is anindolylmethyl group, except compounds in which R₂ is a methyl group..Iadd.31. The compound according to claim 30, in which R₁ is selectedfrom the group which consists of an alkyl group having 1 to 12 carbonatoms; a 5-, 6- or 7-membered alicyclic alkyl group; and an alkyl grouphaving 1 to 5 carbon atoms substituted by an aromatic hydrocarbon grouphaving 6 to 12 carbon atoms. .Iaddend..Iadd.32. The compound accordingto claim 31, in which said alkyl group having 1 to 12 carbon atoms isisoamyl or cyclohexylmethyl, and said alkyl group having 1 to 5 carbonatoms substituted by the aromatic hydrocarbon group is phenylethyl,naphthylmethyl or naphthylethyl. .Iaddend..Iadd.33. The compoundaccording to claim 30, in which said indolylmethyl group isindol-3-ylmethyl. .Iaddend..Iadd.34. The compound according to claim 30,in which R₂ is (i) a C₃₋₈ alkyl group, (ii) a cyclohexylmethylene groupor (iii) a benzyl group; and R₃ is an indolylmethyl group..Iaddend..Iadd.35. A method for bringing about endothelin-convertingenzyme inhibiting activity in a warm-blooded animal, which comprisesadministering an effective amount of the compound or thepharmaceutically acceptable salt thereof claimed in claim 30 to thewarm-blooded animal. .Iaddend..Iadd.36. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and the compound ofclaim 30 or a pharmaceutically acceptable salt thereof..Iaddend..Iadd.37. A method for bringing about endothelin-convertingenzyme inhibiting activity in a warm-blooded animal, which comprisesadministering to the animal an effective amount of a compoundrepresented by the formula I! or a pharmaceutically acceptable saltthereof: ##STR12## wherein: R₁ is (i) a C₁₋₈ alkyl group which may besubstituted by C₅₋₈ cycloalkyl, halogen, hydroxy which may be protected,C₁₋₂ alkoxy, keto or amino which may be protected, (ii) a 5 to 7 membercycloalkyl group which may be substituted by lower alkyl having 1 to 3carbon atoms, halogen, hydroxy which may be protected C₁₋₂ alkoxy, keto,or amino which may be protected or (iii) an aralkyl group which may besubstituted by lower alkyl having 1 to 3 carbon atoms, C₅₋₆ cycloalkyl,halogen, hydroxy which may be protected, or C₁₋₂ alkoxy;R₂ is (i) a C₁₋₈alkyl group (ii) a cyclohexylmethyl group or (iii) a benzyl group; andR₃ is an indolylmethyl group or a benzyl group; except compounds inwhich (i) R₂ is a methyl group and (ii) R₁ isbenzyloxgycarbonylaminomethyl, R₂ is isobutyl and R₃ is benzyl..Iaddend.